The repertoire of substances available for therapeutic purposes consists primarily of relatively low-molecular weight organic compounds. Recently, the repertoire has been expanded to include proteinaceous materials which have been engineered for efficacy, specificity and stability. Increasing attention is now being focused on the therapeutic potential of other classes of biomacromolecules, including nucleic acids.
Nucleic acids are linear phosphopentose polymers bearing pendant adenine (A), guanine (G), cytosine (C), and thymine (T) [or the related uracil (U)] base groups. The pentose may be ribose (RNA) or 2'deoxyribose (DNA). They are attractive candidates for therapeutics due to the high potential for selectivity. The basis for this high selectivity is the well-known ability of a nucleic acid to form an antiparallel, two-stranded, helical structure (or duplex) with its structural complement through the formation of hydrogen bonds between the bases on opposite strands (Watson-Crick base pairs). Complementarity is defined as the pairing of G with C and A with T [or U] on opposite strands. Duplexes with perfect complementarity are thermodynamically preferred. For short [&lt;20 residues or nucleotides (nt)] oligonucleotides, a single improper pairing or mismatch can significantly destabilize the duplex. Thus one can, in principle, selectively address a single site in a 3.times.10.sup.9 nt human genome (the genetic material of a human in its entirety) with an oligonucleotide of 16-20 nt. This is substantially greater selectivity than one can generally achieve with traditional, low-molecular weight agents. With this degree of potential selectivity, one can consider the approach of exerting a therapeutic effect at the level of gene expression. For viral agents which act through integration of their genetic material into the host system, one can envision blocking one of the many steps involved in integration and replication.
Most of the attempts to use nucleic acids as complementary addressed therapeutic agents have involved single-stranded targets. Such targets include messenger RNA (mRNA) and single-stranded viral genomes. In such cases, the reagent nucleic acids are complementary to the target and are referred to as "anti-sense reagents". The process of using such agents to exert a specific effect is referred to herein as "anti-sense targeting".sup.1-3,32.
More recently, a second, high-specificity mode of nucleic acid binding has been investigated. It has been found that certain sequences of duplex DNA will bind a third strand to form a triple helix or triplex.sup.4. Triplex formation involves the formation of base triples with the additional base forming hydrogen bonds in the so-called Hoogsteen mode. Reagents designed to bind in such a mode are referred to herein as "triplex reagents" and the process of using such reagents to exert a specific effect will be referred to as "triplex targeting". The advantage of the triplex targeting approach is that one can address double-stranded genomic DNA directly. The disadvantage is that, at least at this time, not all sequences can be addressed in this fashion.
The ease of synthesis and chemical stability of simple, unmodified oligodeoxynucleotides has led to widespread investigation of their use as anti-sense reagents. This approach has been used with varying degrees of success in vitro against human immunodeficiency virus (HIV), Rous sarcoma virus, and c-myc oncogene expression, among others..sup.28
Simple oligodeoxynucleotide anti-sense reagents may exert their effects in one or both of two ways. They can simply bind their target through duplex formation thereby reducing the available concentration of functional single-stranded target. Alternatively, in the case where the target is a single-stranded RNA, the RNA/DNA hybrid may Serve as a substrate for endogenous ribonuclease H (RNaseH). RNaseH is an enzyme which will cleave the RNA strand of an RNA/DNA hybrid through phosphodiester bond hydrolysis. The mediation of RNaseH can allow anti-sense reagents to operate at concentrations well below those required to drive all of the target to hybrid since the reagent itself is not cleaved and each molecule can direct the cleavage of many molecules of target.
A number of structural modification approaches to improve the function of oligodeoxynucleotides as anti-sense reagents have been investigated. One class of modification involves the attachment of chemical appendages to the reagent to stabilize the reagent/target duplex or cleave the target at the site of attachment. Acridine derivatives attached via flexible tethers have been shown to improve the thermodynamic stability of the duplex through intercalation.sup.5. Similarly, oligodeoxynucleotides bearing tethered psoralens can be covalently cross-linked to target following irradiation of the duplex.sup.6. Cross-linking can also be accomplished through the use of tethered alkylating agents.sup.7. Cleavage of the target through the use of oligodeoxynucleotides bearing tethered ethylenediaminetetraacetic acid (EDTA)/iron.sup.8 or 1,10-phenanthroline/copper.sup.9 has been demonstrated in vitro. Numerous other attachments for these purposes have been described. Functionalization with poly (L-lysine) has been employed to improve transport..sup.31
The use of oligodeoxynucleotides as anti-sense reagents in vivo is hampered, however, by two fundamental problems. The first problem is that small single-stranded oligodeoxynucleotides are rapidly digested by endogenous nucleases. As a result high in vivo concentrations are difficult to sustain. The second problem is that oligodeoxynucleotides are highly charged having roughly one full negative charge per nucleotide residue. This generally results in a reduced rate of transport across membranes which in some cases limits the access to the ultimate site of action. These two effects combine to afford relatively low bioavailability.
The attachment of chemical functionalities (as described above) to the terminii of oligodeoxynucleotides can provide enhanced nuclease-resistance in some cases. Alpha-Oligodeoxynucleotides, in which the attachment of the base to the ribofuranosyl ring has been changed from beta- to alpha-, form parallel stranded duplexes and show increased nuclease resistance.sup.10.
In several approaches to solve the stability and transport problems, the central phosphorus atom in the linkage has been retained but attached atoms have been replaced or modified. O-Alkylphosphotriesters are uncharged but are more bulky than the natural linkage and show somewhat reduced chemical stability.sup.11,12. Phosphorothioate diesters are isostructural with the natural linkage and show increased resistance to nucleases but are still charged.sup.13. Methylphosphonates are uncharged and considerably more lipophilic but fully replaced hybrids are not substrates of RNaseH.sup.14.
One fundamental difficulty with these analogues is the fact that all derive from a single replacement of one of the non-bridging oxygen atoms on the phosphodiester linkage. Since that phosphorus is prochiral within the linkage, non-specific replacement of one oxygen results in the formation of a chiral center and hence a pair of diastereomers. Each additional non-specific replacement doubles the number of diastereomers present. These diastereomers have differing physical properties complicating analysis and in some cases have been shown to have widely differing abilities to form hybrids.sup.34. Thus oligodeoxynucleotide analogues with multiple linkage replacements are generally complex mixtures of species which can have widely differing biological efficacy.
The diastereomer mixture problem can be circumvented by replacement of both bridging oxygens in the phosphodiester linkage with the same chemical group. Along these lines, phosphorodithioate diesters have been investigated.sup.15. However, like the phosphorothioate linkage, this moiety is still charged.
Approaches involving more extensive modifications have also been reported. Replacement of the phosphodiester linkage with a carbon centered, neutral carbamate have been investigated.sup.16,17. The planarity of the carbamate linkage ensures that its introduction does not generate diastereomers but it represents a departure from tetrahedral geometry of the phosphodiester with consequences that have not been fully explored. At least one derivative has been shown to be able to form duplex with complementary target. The carbamate's nuclease sensitivity and ability to activate RNaseH have not yet been reported on.
Several recent studies have focused on oligodeoxynucleotides with partial phosphodiester linkage replacement. The rationale here is that one may be able to engineer a balanced profile of desirable properties (e.g., nuclease resistance, hybrid stability, RNaseH activation, etc.). These studies while useful are unlikely to lead to a general solution to the problems listed above.
With some analogues, the situation is further complicated by the observation that biological effects are being exerted in a non-sequence-specific manner.sup.18,19. The origins of these effects remain obscure.
The described list of modifications explored in attempts to enhance the function of oligodeoxynucleotides as anti-sense reagents is representative but is by no means exhaustive. Two recent reviews.sup.1,2 and one monograph.sup.32 dealing with this subject area are comprehensive.
A series of naturally occurring sulfamoyl mononucleoside antibiotics and some synthetic analogs have been described.sup.21-24. In addition, analogs of DNA containing sulfides, sulfoxides and sulfones as linking groups between subunits capable of forming bonds with natural oligonucleotides have been described..sup.33 The .beta.-decay of P.sup.32 oligonucleotides is expected to give a sulfate linkate oligonucleotide; however, such sulfate linked oligonucleotides have not been reported.
Applicants have developed a novel series of compounds in which one or more of the internucleotide phosphodiester linkages in oligonucleotide analogs have been replaced by a sulfur-based linkage. This linkage is isostructural and isoelectronic with the phosphodiester. Applicants have found the linkage to be synthetically accessible, chemically robust, nuclease resistant, and capable of supporting duplex formation. In addition, such compounds would have potential as antiviral agents and utility as hybridization probes.